Therapeutic compositions and screening methods relating to 3&#39; -tyrosyl-DNA-phosphodiesterase

ABSTRACT

The invention provides a compound that inhibits the activity of 3′-tyrosyl-DNA phophodiesterase (TDP). In one embodiment, the compound is a polynucleotide-3′-bridging phosphoramidate. The invention also provides a method of inhibiting TDP activity. In one embodiment, the method involves contacting the enzyme with a polynucleotide-3′-bridging phosphoramidate, polynucleotide-3′-alkyl phosphonate, polynucleotide-3′-alkyl phosphotriester, polynucleotide-bridging-alkyl phosphonate, nucleotide-3′-bridging phosphoramidate, nucleotide-3′-alkyl phosphonate, nucleotide-3′-alkyl phosphotriester, or nucleotide-bridging-alkyl phosphonate. The invention further provides a method of decreasing cellular proliferation. The method involves contacting a TDP-containing cell with an effective amount of a TDP inhibiting compound sufficient to inhibit TDP activity in said cell. In addition, the invention provides a method of screening for compounds that modulate the activity of TDP.

[0001] This application is based on, and claims the benefit of, U.S. Provisional Application No. 60/311,058, filed Aug. 8, 2001, which is incorporated herein by reference.

SUMMARY OF THE INVENTION

[0002] The invention provides a compound that inhibits the activity of 3′-tyrosyl-DNA phophodiesterase (TDP). In one embodiment, the compound is a polynucleotide-3′-bridging phosphoramidate. The invention also provides a method of inhibiting TDP activity. In one embodiment, the method involves contacting the enzyme with a polynucleotide-3′-bridging phosphoramidate, polynucleotide-3′-alkyl phosphonate, polynucleotide-3′-alkyl phosphotriester, polynucleotide-bridging-alkyl phosphonate, nucleotide-3′-bridging phosphoramidate, nucleotide-3′-alkyl phosphonate, nucleotide-3′-alkyl phosphotriester, or nucleotide-bridging-alkyl phosphonate.

[0003] The invention further provides a method of decreasing cellular proliferation. The method involves contacting a TDP-containing cell with an effective amount of a TDP inhibiting compound sufficient to inhibit TDP activity in said cell.

[0004] In addition, the invention provides a method of screening for compounds that modulate the activity of TDP. The method consists of contacting TDP, or a fragment or modification thereof having TDP activity, with a compound under conditions that allow TDP activity, determining an amount of TDP activity, and identifying a compound that modulates TDP activity. The compounds so identified are potentially useful therapeutic agents for decreasing cell proliferation.

BACKGROUND OF THE INVENTION

[0005] Cancer is currently the second leading cause of mortality in the United States. However, it is estimated that by the year 2000 cancer will surpass heart disease and become the leading cause of death in the United States. Cancerous tumors result when a cell escapes from its normal growth regulatory mechanisms and proliferates in an uncontrolled fashion. Tumor cells can metastasize to secondary sites if treatment of the primary tumor is either not complete or not initiated before substantial progression of the disease. Early diagnosis and effective treatment of tumors is therefore essential for survival.

[0006] Continuous developments over the past quarter century have resulted in substantial improvements in the ability of a physician to diagnose a cancer in a patient. Unfortunately, methods for treating cancer have not kept pace with those for diagnosing the disease. Thus, while the death rate from various cancers has decreased due to the ability of a physician to detect the disease at an earlier stage, the ability to treat patients presenting more advanced disease has progressed only minimally.

[0007] A hurdle to advances in treating cancer is the relative lack of agents that can selectively target the cancer, while sparing normal tissue. For example, radiation therapy and surgery, which generally are localized treatments, can cause substantial damage to normal tissue in the treatment field, resulting in scarring and, in severe cases, loss of function of the normal tissue. Chemotherapy, which generally is administered systemically, can cause substantial damage to organs such as bone marrow, mucosae, skin and the small intestine, which undergo rapid cell turnover and continuous cell division. As a result, undesirable side effects, for example, nausea, hair loss and reduced blood cell counts, occur as a result of systemically treating a cancer patient with chemotherapeutic agents. Such undesirable side effects often limit the amount of a treatment that can be administered.

[0008] A related hurdle prohibiting significant advances in cancer treatment is the relative lack of cancer specific targets. For example, treatments have been attempted where cytotoxic agents have been directed to tumor cell surface markers. However, such approaches similarly result in pleiotropic side effects and damage to normal tissue because of the lack of availability or specificity of tumor cell markers. Due to such shortcomings in treatment, cancer remains a leading cause of patient morbidity and death.

[0009] Thus, there exists a need for improved methods of treating cancer and other proliferative pathological conditions. The present invention satisfies this need and provides related advantages as well.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows PAGE analysis of purified 3′-tyrosyl-DNA-phosphodiesterase.

[0011]FIG. 2 shows HPLC analysis of 3′-derivatized polynucleic acids.

[0012]FIG. 3 shows 3′-tyrosyl-DNA-phosphodiesterase activity.

[0013]FIG. 4 shows kinetic analysis of 3′-tyrosyl-DNA-phosphodiesterase. FIG. 4A shows the velocity of cleavage of 3′-(4′-nitro-phenyl)-oligonucleotides plotted as a function of substrate concentration. FIG. 4B shows an Eadie-Hofstee plot of the data presented in FIG. 4A.

[0014]FIG. 5 shows an exemplary synthesis scheme for oligonucleotides containing a 3′-phospho-phenyl group in which the phenyl moiety is post-synthetically derivatized.

[0015]FIG. 6 shows a comparison of two TDP reaction mechanisms.

[0016]FIG. 7 shows a comparison of mechanism based inhibitors.

[0017]FIG. 8 shows an exemplary synthetic combinatorial library.

[0018]FIG. 9 shows a scheme for deconvoluting a combinatorial library.

[0019]FIG. 10 shows the chemical structures of a 3′-tyrosyl-DNA-phosphodiesterase substrate and the corresponding product, the formation of which can be monitored spectrophotometrically.

[0020]FIG. 11 shows the chemical structures of a 3′-tyrosyl-DNA-phosphodiesterase substrate and the corresponding product, the formation of which can be monitored in a fluorescence-based assay.

[0021]FIG. 12 shows an alignment of the amino acid sequences of TDP polypeptide sequences from drosphila melanogasler (SEQ ID NO:1), mouse (SEQ ID NO:2), human (SEQ ID NOS:3 and 4), C.elegans (SEQ ID NO:5) and S.cerevisiae (SEQ ID NO:6)(Pouliot, et al. Science, 286:552-555 (1999)).

DETAILED DESCRIPTION OF THE INVENTION

[0022] This invention is directed to inhibitors of 3′-tyrosyl-DNA phosphodiesterase (TDP) activity. TDP is an enzyme that cleaves the chemical bond between a topoisomerase and a DNA molecule to which topoisomerase is bound. Topoisomerases are cellular enzymes that function by breaking the DNA backbone, allowing DNA to undergo topological change, and then resealing the break. During this process, topoisomerases form a covalent bond with the DNA prior to the resealing step. Under circumstances in which the resealing step fails, topoisomerase remains covalently bound to the DNA. Such topoisomerase-DNA complexes disrupt normal cellular replication, leading to cell death. For example, DNA mismatches, nicked DNA, camptothecin-like drug induced- and topoisomerase-induced mutation have been shown to cause covalent complexes to accumulate in vitro (Yeh et al. J. Biol. Chem., 269:15498-15405 (1994), Lanza et al. J. Biol. Chem 271:6989-6986 (1996)). TDP is a repair enzyme that prevents the accumulation of these faulty covalent topoisomerase-DNA complexes that normally occur during the life time of a cell. Inhibiting TDP activity thus promotes cell death by allowing the accumulation of topoisomerase-DNA complexes. Therefore, inhibitors of TDP provide a means of destroying unwanted cells, such as pathologically aberrant cells associated with proliferative diseases, infectious disease and other disorders of excessive or unwanted cell proliferation.

[0023] In one embodiment, the invention is directed to compounds that inhibit TDP activity. Such compounds include nonhydrolyzable analogs, such as derivatized polynucleotides, which act as competitive inhibitors of TDP activity. The compounds of the invention are useful for inhibiting TDP contained in a variety of samples, as well as in cells, tissues and organs of an animal, including a human.

[0024] As used herein, the term “polynucleotide-3′-bridging phosphoramidate” is intended to mean a polynucleotide that contains at least one “3′-bridging phosphoramidate moiety.” As used herein, the term “3′-bridging phosphoramidate moiety” is intended to mean a nucleotide having at least one phosphate group linked by a phosphodiester bond at the 3′ position of the sugar ring, the phosphate group having a phosphate oxygen substituted by nitrogen. The nitrogen can be bound to a chemical group, in particular a chemical group that would result in the formation of a poor leaving group in a reaction in which the nitrogen atom is attacked by a nucleophile. The nitrogen atom forms a bridge or link between the bound phosphorus atom and a bound chemical group.

[0025] A “polynucleotide-3′-bridging phosphoramidate” is a compound having the following structure:

[0026] wherein, X is a nucleotide; y is a positive integer; R1 is any nucleotide base; R2 is H, OH, halo, amino, alkyl or azido; and R3 is phenyl, aryl, substituted phenyl or substituted aryl.

[0027] A “nucleotide-3′-bridging phosphoramidate” is a compound having the following structure:

[0028] wherein, X is a nucleotide; y is a positive integer; R1 is any nucleotide base; R2 is H, OH, halo, amino, alkyl or azido; and R3 is phenyl, aryl, substituted phenyl or substituted aryl; and R4 is a hydrogen atom, hydroxyl, azido, halo, amino or O-alkyl.

[0029] As used herein, the term “polynucleotide-3′ phosphorothioate” is intended to mean a polynucleotide that contains at least one 3′-phosphorothioate moiety. As used herein, the term “3′-phosphorothioate moiety” is intended to mean a nucleotide having at least one phosphate group linked by a phosphodiester bond at the 3′ position of the sugar ring, the phosphate group having a phosphate oxygen substituted by sulfur.

[0030] A “polynucleotide-3′ phosphorothioate” is a compound having the following structure:

[0031] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; R3 is oxygen; and R4 is a halo, alkyl, substituted alkyl, phenyl or substituted phenyl.

[0032] An exemplary polynucleotide-3′ phosphorothioate contains a phenyl moiety at the R₄ position, such as:

[0033] A “nucleotide-3′ phosphorothioate” is a compound having the following structure:

[0034] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo, amino, alkyl or azido; R3 is oxygen; R4 is a halo, alkyl, substituted alkyl, phenyl or substituted phenyl; and R5 is a hydrogen atom, hydroxyl, azido, amino, halo, or O-alkyl.

[0035] As used herein, the term “polynucleotide-3′-alkyl phosphonate” is intended to mean a polynucleotide that contains at least one 3′-alkyl phosphonate moiety. As used herein, the term “3′-alkyl phosphonate moiety” is intended to mean a nucleotide having at least one phosphate group linked by a phosphodiester bond at the 3′ position of the sugar ring, the phosphate group having a phosphate oxygen substituted by an alkyl group, such as a methyl or ethyl group and a phosphate oxygen substituted by another moiety, R3.

[0036] A “polynucleotide-3′ alkyl phosphonate” is a compound having the following structure:

[0037] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; R3 is a halo, alkyl, substituted alkyl, phenyl or substituted phenyl; and R4 is alkyl.

[0038] An exemplary polynucleotide-3′ alkyl phosphonate contains a phenyl moiety at the R3 position, such as:

[0039] A nucleotide-3′ alkyl phosphonate is a compound having the following structure:

[0040] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; R3 is halo, alkyl, substituted alkyl, phenyl or substituted phenyl; R4 is alkyl; and R5 is a hydrogen atom, hydroxyl, azido, halo, amino, or O-alkyl.

[0041] As used herein, the term “polynucleotide-3′-alkyl phosphotriester” is intended to mean a polynucleotide that contains at least one 3′-alkyl phosphotriester moiety. As used herein, the term “3′-alkyl phosphotriester moiety” is intended to mean a nucleotide having at least one phosphate group linked by a phosphodiester bond at the 3′ position of the sugar ring, the phosphate group having a phosphate oxygen substituted by a phosphoester group.

[0042] A “polynucleotide-3′-alkyl phosphotriester” has the following structure:

[0043] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; and R3 is a halo, alkyl, substituted alkyl, phenyl or substituted phenyl.

[0044] A “nucleotide-3′-alkyl phosphotriester” has the following structure:

[0045] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; R3 is a halo, alkyl, substituted alkyl, phenyl or substituted phenyl; and R4 is a hydrogen atom, hydroxyl, azido, halo, amino, or O-alkyl.

[0046] As used herein, the term “polynucleotide-bridging-alkyl phosphonate” is intended to mean a polynucleotide that contains at least one bridging-alkyl phosphonate moiety. As used herein, the term “bridging-alkyl phosphonate moiety” is intended to mean a nucleotide having at least one phosphate group linked by a phosphodiester bond at the 3′ position of the sugar ring, the phosphate group having a phosphate oxygen substituted by a substituted alkyl group.

[0047] A “polynucleotide-bridging-alkyl phosphonate” has the following structure:

[0048] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; R3 is substituted alkyl.

[0049] An exemplary “polynucleotide-bridging-alkyl phosphonate” is:

[0050] A “nucleotide-bridging-alkyl phosphonate” has the following structure:

[0051] wherein, X is a nucleotide; y is a positive integer; R1 is a nucleotide base; R2 is a hydrogen atom, hydroxyl, halo amino, alkyl or azido; R3 is substituted alkyl; and R4 is a hydrogen atom, hydroxyl, azido, halo, amino, or O-alkyl.

[0052] As used herein, the term “polynucleotide” is intended to mean a chain of two or more nucleotide 5′-monophosphate residues linked through one or more phosphodiester bonds. A nucleotide of a polynucleotide can contain a variety of glycose moieties, such as, for example, D-ribose and D-2-deoxyribose, as well as modified glycose moieties such as cytarabine. Therefore, a polynucleotide encompasses ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), a hybrid of RNA and DNA, as well as RNA and DNA molecules containing nucleotides which have modified glycose moieties.

[0053] A nucleotide, including a nucleotide contained in a polynucleotide, can contain any nucleic acid base, including both naturally occurring and modified bases. Examples of naturally occurring bases include guanine, adenine, thymine, cytosine and uridine. Examples of modified bases include bases that are detectable by a variety of analytical methods, for example, fluorescent bases and bases with useful absorbance characteristics. Exemplary modified bases include 4-thio-uridine, pseudouridine, 2′-deoxy-uridine, 5-fluoro-uridine, 5-bromo-uridine, 5-iodo-uridine, 2′-amino-uridine, 2′-fluoro-uridine, 2′-fluoro-cytidine, 2′-amio-butyryl-pyrene-uridine, 5-fluoro-cytidine, ribo-thymidine, 5-methyl-cytidine, inosine, purine ribonucleoside, 2-aminopurine, 2,6-diaminopurine, N3-methyl uridine and ribavirin. A variety of other structures can be incorporated into a synthetic base. Particularly useful structures include those that render a molecule detectable, favorably alter an activity of the molecule or function as purification tags. For example, a base can contain 3′ and 5′ modifications such as 3′-puromycin, 3′-inverted deoxy thymidine, 3′-thioate linkage, 5′-biotin, a fluorescent moiety such as 5′-fluorescein, 5′-Cy3, 5′-Cy5, 5′-tetrachloro-fluorescein, and other moieties, with and without atomic spacers.

[0054] A nucleotide or polynucleotide can be naturally occurring or synthetically produced. For example, a polynucleotide can be isolated from an organism or synthesized using various methods, such as automated methods well known in the art. A naturally occurring polynucleotide can be, for example, an RNA such as an mRNA, a DNA such as a cDNA or genomic DNA, and can represent the sense strand, the anti-sense strand, or both.

[0055] A polynucleotide can be a single stranded, duplex or branched polynucleotide. A duplex polynucleotide is a polynucleotide having two strands associated together by hydrogen bonding. A strand of a duplex polynucleotide can contain one or more mismatched, absent or additional nucleotides that do not associate with the cognate nucleotide in the partner strand, so long as the duplex remains associated. A duplex polynucleotide includes polynucleotides of both synthetic and natural origin. Thus, a duplex polynucleotide can be, for example, cDNA, genomic DNA, RNA, mRNA, synthetic DNA, including, for example, annealed complementary oligonucleotides or polynucleotides. A polynucleotide of natural origin can be derived from any eukaryotic, prokaryotic or viral source. Duplex DNA can have blunt ends, 3′ terminal end overhangs and 5′ terminal end overhangs. Single stranded, duplex or branched DNA further can contain a tag or moiety, such as a tag useful for detection or purification.

[0056] The term “alkyl” denotes such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, amyl, tert-amyl, hexyl and the like. A preferred alkyl group is methyl.

[0057] The term substituted alkyl groups is intended to mean alkyl groups, such as C1 to C6 and C7 to C12 alkyl groups that are substituted by one or more halogen, hydroxy, protected hydroxy, oxo, protected oxo, cyclohexyl, naphthyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, guanidino, heterocyclic ring, substituted heterocyclic ring, imidazolyl, indolyl, pyrrolidinyl, C1 to C7 alkoxy, C1 to C7 acyl, C1 to C7 acyloxy, nitro, C1 to C7 alkyl ester, carboxy, protected carboxy, carbamoyl, carboxamide, protected carboxamide, N-(C1 to C6 alkyl)carboxamide, protected N-(C1 to C6 alkyl)carboxamide, N,N-di(C1 to C6 alkyl)carboxamide, cyano, methylsulfonylamino, thio, C1 to C4 alkylthio or C1 to C4 alkyl sulfonyl groups. The substituted alkyl groups can be substituted once or more with the same or with different substituents.

[0058] Examples of the above substituted alkyl groups include the chloromethyl, bromomethyl, iodomethyl, trifluoromethyl, 6-hydroxyhexyl, 2,4-dichloro(n-butyl), 2-aminopropyl, chloroethyl, bromoethyl, fluoroethyl, iodoethyl, chloropropyl, bromopropyl, fluoropropyl, iodopropyl and the like.

[0059] The term “substituted phenyl” specifies a phenyl group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, C1 to C6 alkyl, C1 to C7 alkoxy, C1 to C7 acyl, C1 to C7 acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(C1 to C6 alkyl)carboxamide, protected N-(C1 to C6 alkyl)carboxamide, N, N-di(C1 to C6 alkyl)carboxamide, trifluoromethyl, N-((C1 to C6 alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, substituted or unsubstituted, such that, for example, a biphenyl results.

[0060] Examples of the term “substituted phenyl” include a mono- or di(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl and the like; a mono or di(hydroxy)phenyl group such as 2, 3 or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 2, 3 or 4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl; a mono- or di(alkyl)phenyl group such as 2, 3 or 4-methylphenyl, 2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3 or 4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono or di(alkoxyl)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or 4-methoxyphenyl, 2, 3 or 4-ethoxyphenyl, 2, 3 or 4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2, 3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono-or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3, or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or 4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl” represents disubstituted phenyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy 4-chlorophenyl and the like.

[0061] The terms “halo” and “halogen” refer to fluoro, chloro, bromo or iodo groups.

[0062] As used herein, the term “3′-tyrosyl-DNA-phosphodiesterase” or “TDP,” refers to a class of enzymes that hydrolyze a 3′-tyrosyl phosphodiester, such as a 3′-tyrosyl phosphodiester bond between a topoisomerase and the 3′ end of a polynucleotide. An exemplary TDP is yeast TDP. Nucleotide and amino acid sequences of yeast TDP are available in the GenBank database as accession number Z36092.1. EST sequences corresponding to TDP genes of other organisms, including human, mouse, Drosophila melanogaster and Caenorhabditis elegans are available in the GenBank database. For example, ESTs corresponding to human TDP include AA477148, AA489121 and AI480141. An exemplary human TDP nucleotide sequence (SEQ ID NO:7), which encodes amino acid sequence SEQ ID NO:8 is available at GenBank accession number NM_(—)018319.

[0063] TDP functions to cleave a covalent 3′-tyrosyl phosphodiester bond between a topoisomerase and a polynucleotide. Therefore, binding to a topoisomerase-polynucleotide complex is a “TDP activity.” Another “TDP activity” is cleavage of a 3′-tyrosyl phosphodiester bond, such as a 3′-tyrosyl bond between topoisomerase and a polynucleotide. As used herein, the term “TDP enzymatic activity” is intended to mean both binding of TDP to a topoisomerase-polynucleotide complex and cleavage of a 3′-tyrosyl bond between topoisomerase and a polynucleotide.

[0064] The term TDP encompasses native TDP from a variety of species, and also encompasses polypeptides containing minor modifications of a native TDP sequence, and fragments of a full-length native TDP, so long as the modified polypeptide or fragment retains one or more biological activities of a native TDP, such as the abilities to bind to topoisomerase-DNA complexes, and cleave a 3′-tyrosyl phosphodiester bond. A modification of a TDP can include additions, deletions, or substitutions of amino acids, so long as a biological activity of a native TDP is retained. For example, a modification can serve to alter the stability or activity the polypeptide, or to facilitate its purification.

[0065] A modified TDP can contain amino acid analogs, derivatives and mimetics. Such modifications and functional equivalents of amino acids are well known to those skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivitization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics arginine would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the ε-amino group of the side chain of the naturally occurring amino acid. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0066] A TDP can be modified, for example, to increase polypeptide stability, alter a TDP activity, facilitate detection or purification, or render the enzyme better suited for a particular application, such as by altering substrate specificity. Computer programs known in the art can be used to determine which amino acid residues of a TDP can be modified as described above without abolishing a TDP activity (see, for example, Eroshkin et al., Comput. Appl. Biosci. 9:491-497 (1993)). In addition, structural and sequence information can be used to determine the amino acid residues important for TDP activity. For example, a TDP crystal structure (Davies et al. Structure 19(2):237-248 (2002)) and comparisons of TDP amino acid sequences, such as that shown in FIG. 12 can provide guidance in determining amino acid residues that can be altered without abolishing TDP activity.

[0067] A “fragment” of a TDP is intended to mean a portion of a TDP that retains a portion of the activity of a native TDP, and can retain at least about the same activity as a native TDP. A fragment of a TDP can contain a modification. It is understood that the activity of a TDP can be measured by the methods disclosed herein.

[0068] The term “TDP” includes native or recombinantly expressed TDP expressed in cells or cell lysates, and includes chemically synthesized TDP. A TDP can contain an exogenous amino acid sequence, such as, for example, a tag that facilitates purification or identification. Exemplary tags include histidine tags, glutathione-S transferase tags, FLAG tags and myc tags. Other chemical tags such as biotin and fluorescent or radioactive tags can be present on a TDP polypeptide or nucleic acid molecule.

[0069] As used herein, the term “TDP modulating” when used in reference to a compound is intended to mean that the compound alters the amount or rate of a TDP activity, such as a binding interaction between TDP and a topoisomerase-polynucleotide complex or 3′-tyrosyl phosphodiesterase activity, compared to a reference level of TDP activity. A TDP modulating compound can thus be, for example, a compound that selectively binds TDP, a compound that modulates an interaction between TDP and a substrate, such as a topoisomerase-polynucleotide complex or derivatized polynucleotide, a compound that modulates 3′-tyrosyl phophodiesterase activity, or a compound with more than one of these effects. A TDP modulating compound can increase or decrease the amount or rate of a TDP activity. A TDP modulating compound can act directly or indirectly. A TDP modulating compound that acts directly can, for example, bind to a TDP and alter substrate affinity, substrate specificity, or susceptibility to proteolysis. A TDP modulating compound that acts indirectly can, for example, bind to a molecule that regulates the expression level or activity of TDP, thereby altering the amount or rate of TDP activity. Therefore, TDP modulating compounds include a “TDP inhibiting” compound. As used herein, the term “TDP inhibiting” when used in reference to a compound is intended to mean a compound that decreases, directly or indirectly, the amount or rate of TDP activity. A TDP inhibiting compound can, for example, bind to a TDP and reduce substrate affinity, reduce substrate specificity, compete with a substrate for binding to TDP, or increase susceptibility of TDP to proteolysis.

[0070] As used herein, the term “decreasing” when used in reference to cellular proliferation is intended to mean effecting a reduction in the amount or rate of cell growth. Effecting a reduction in the amount or rate of unregulated cell growth in TDP containing cells is a specific example of decreasing cellular proliferation.

[0071] As used herein, the term “TDP-containing cell” is intended to refer to a cell having a measurable amount of TDP. A TDP-containing cell can contain an amount of TDP that is sufficient to perform normal repair function of TDP.

[0072] As used herein, the term “reducing the severity,” when used in reference to a proliferative disease, is intended to mean an arrest or decrease in clinical symptoms, physiological indicators or biochemical markers of proliferative disease. Clinical symptoms include perceptible, outward or visible signs of disease. Physiological indicators include detection of the presence or absence of physical and chemical factors associated with a process or function of the body. Biochemical markers include those signs of disease that are observable at the molecular level, such as the presence of a disease marker, such as a tumor marker. A tumor marker is a substance in the body that usually indicates the presence of cancer. Tumor markers are usually specific to certain types of cancer and are usually found in the blood or other tissue samples. One skilled in the art will be able to recognize specific clinical symptoms, physiological indicators and biochemical markers associated with a particular proliferative disease.

[0073] As used herein, the term “effective amount” when used in reference to reducing the severity of a proliferative disease, such as cancer, is intended to mean an amount of TDP inhibiting compound administered to an individual required to effect a decrease in the amount or rate of spread of a neoplastic condition or pathology. The amount of a TDP inhibiting compound required to be effective will depend, for example, on the type or types of TDP inhibiting compounds administered, the pathological condition to be treated and the level of abundance of TDP in a cell, as well as the weight and physiological condition of the individual, and previous or concurrent therapies. An amount considered as an effective amount for a particular application of TDP inhibiting compound will be known or can be determined by those skilled in the art, using the teachings and guidance provided herein. One skilled in the art will recognize that the condition of the patient can be monitored throughout the course of therapy and that the amount of the modulating compound that is administered can be adjusted according to the individual's response to therapy.

[0074] As used herein, the term “cancer” is intended to mean a class of diseases characterized by the uncontrolled growth of aberrant cells, including a variety of known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue or solid tumor. Exemplary specific cancers include breast, ovarian, small-cell, leukemia, lymphoma, melanoma and prostate cancers.

[0075] The invention provides a TDP inhibiting compound. The structure of the compound is:

[0076] wherein,

[0077] X is a nucleotide;

[0078] Y is a positive integer;

[0079] R1 is any nucleotide base;

[0080] R2 is H; and

[0081] R3 is phenyl or substituted phenyl.

[0082] The polynucleotide-3′-bridging phosphoramidate of the invention can contain a variety of substituents that preferably function as poor leaving groups at the R3 position.

[0083] Exemplary polynucleotide-3′-phosphodiester derivatives and k_(rel) values representing the rates of cleavage by human TDP, relative to 3′-paranitrophenyl, indicated in parentheses, include: aniline derivative (k_(rel) = 0.05)

toluidine derivative (k_(rel) = 0.025)

N,N-dimethyl-1,4- phenylene-diamine derivative (k_(rel) = 0.0125)

Azidoaniline derivative (undetectable activity)

[0084] Other exemplary moieties which can be present in a polynucleotide-3′-bridging phosphoramidate, polynucleotide-3′alkylaphosphonate, polynucleotide 3′-phosphorothioate, polynucleotide-3′-alkyl phosphotriester, polynucleotide-bridging-alkyl phosphonate, nucleotide-3′-bridging phosphoramidate, nucleotide-3′-alkyl phosphonate, nucleotide-3′-alkyl phosphotriester, or nucleotide-bridging-alkyl phosphonate include:

[0085] A polynucleotide-3′-bridging phosphoramidate of the invention can be conveniently prepared as described in Example III, by modifications of methods that produce the polynucleotide-3′ phosphodiester derivative compound of the invention, and by other methods which can be determined by those skilled in the art. Such modifications of the procedure for preparing a polynucleotide-3′phosphodiester derivative can include, for example, the use of alternate solvents, buffers and temperature conditions.

[0086] Methods for preparing a variety of polynucleotide-3′ derivatives are well known to those skilled in the art. Therefore, those skilled in the art will be able to determine methods for producing various polynucleotide-3′ phosphodiester derivatives, polynucleotide-3′ phosphoramidates, polynucleotide-3′-bridging phosphorothioates, polynucleotide-3′-alkyl phosphonates, polynucleotide-3′-alkyl phosphotriesters, polynucleotide-bridging-alkyl phosphonates, nucleotide-3′-bridging phosphoramidates, nucleotide-3′-alkyl phosphonates, nucleotide-3′-alkyl phosphotriesters, and nucleotide-bridging-alkyl phosphonates.

[0087] The invention provides a composition containing a TDP inhibiting compound, such as a polynucleotide-3′-bridging phosphoramidate aniline derivative, and a camptothecin. The term “camptothecin” as used herein is intended to mean a camptothecin or camptothecin derivative that functions as a topoisomerase I inhibitor. Exemplary camptothecins include, for example, topotecan, irinotecan, DX-8951f, SN38, BN 80915, lurtotecan, 9-nitrocamptothecin and aminocamptothesin. A variety of camptothecins have been described, including camptothecins used to treat human cancer patients. Several camptothecins are described, for example, in Kehrer et al., Anticancer Drugs, 12(2):89-105, (2001).

[0088] The invention provides modified nucleotides that inhibit TDP activity. Such modified nucleotides include, for example, nucleotide-3′-bridging phosphoramidates, nucleotide-3′-phosphorothioates, nucleotide-3′-alkyl phosphonates, and nucleotide-3′-alkyl phosphotriesters. Modified nucleotides can be prepared by those skilled in the art using the methods provided herein for preparing various modified polynucleotides. Alternative methods for preparing modified nucleotides are well known to those skilled in the art.

[0089] Exemplary modified nucleotides that inhibit TDP activity are:

[0090] wherein R1 is a hydrogen atom or nucleotide base, R2 is a hydrogen atom, hydroxyl, azido, halo, amino, or O-alkyl; R3 is amino or methyl; R4 is phenyl or substituted phenyl; and R5 is a hydrogen atom, hydroxyl, azido, halo, amino, or O-alkyl.

[0091] The invention provides a method of inhibiting 3′-tyrosyl-DNA-phosphodiesterase (TDP) activity, comprising contacting a TDP with a TDP inhibiting compound, such as a polynucleotide-3′-bridging phosphoramidate aniline derivative.

[0092] A TDP can be contained in a variety of samples, as well as in a cell, tissue or organ of an individual. A TDP can be present in, for example, a fluid or tissue obtained from an animal, a cell obtained from an animal fluid or tissue, cultured cells, recombinant cells or organisms expressing TDP, and lysates or fractions thereof. A TDP can also be contained in a purified preparation.

[0093] The invention provides a method of decreasing cellular proliferation, comprising contacting a TDP-containing cell with an effective amount of a TDP inhibiting compound sufficient to inhibit TDP activity in said cell.

[0094] A TDP inhibiting compound useful in the method of decreasing cellular proliferation can be, for example, a polynucleotide-3′ phosphodiester derivative, polynucleotide-3′-bridging phosphoramidate, a polynucleotide-3′-phosphorothioate, a polynucleotide-3′-alkyl phosphonate, polynucleotide-3′-alkyl phosphotriester, polynucleotide-bridging-alkyl phosphonate, nucleotide-3′-bridging phosphoramidate, nucleotide-3′-alkyl phosphonate, nucleotide-3′-alkyl phosphotriester, nucleotide-bridging-alkyl phosphonate or a combination thereof. Exemplary polynucleotide-3′-bridging phosphoramidates useful in the methods of the invention for decreasing cellular proliferation are polynucleotide-3′-bridging phosphoramidate aniline and toluidine derivatives.

[0095] The methods of the invention for decreasing cell proliferation can be applied to a variety of TDP-containing cells. For example, it can be desirable to decrease cell proliferation in normal or pathologically aberrant cells. Pathologically aberrant cells can be for example, cells having uncontrolled cell growth. Such hyperproliferative cells include, for example, neoplastic cells and cancer cells including ovarian, small-cell, breast, leukemia, lymphoma and other types of cancer cells. A variety of methods can be used to determine if TDP is expressed in a particular cell, such as a cancer cell. Such methods are well known to those skilled in the art and include immunological methods such as ELISA, Western blotting, RIA, immunofluorescence detection methods, methods based on protein or peptide chromatographic separation, methods based on characterization of TDP mRNA and mass spectrometric detection.

[0096] The ability of a TDP modulating compound to alter TDP activity can be determined, for example, using 3′-(4-nitro-phenyl)-oligonucleotides, which provide a convenient calorimetric readout of phosphodiesterase activity, as described in Example II. The effect of a TDP modulating compound on TDP activity can also be tested using 3′-tyrosyl oligonucleotides and phosphorothioate trapped topoisomerase-DNA complexes. A variety of topoisomerase-DNA complexes can be prepared using methods described in Burgin et al., Nuc. Acids Res. 23:29873-2979, (1995), for example. If desired, for cell-based assay of TDP activity, the concentration of topoisomerase-DNA complex can be modulated in a cell, for example, using topoisomerase poisons such as camptothecins, topoisomerase mutants having altered DNA binding characteristics and by altering topoisomerase expression level in a cell.

[0097] Inhibition constants (K_(I)) can be defined by measuring the rate of TDP cleavage as a function of inhibitor concentration. The specificity of the inhibitors can be determined, for example, by assaying the ability of the inhibitors to slow or prevent tyrosine phosphatase activity or topoisomerase I DNA relaxation activity.

[0098] The effect of a TDP inhibiting compound on a neoplastic or cancer cell can be assessed by several criteria well known in the art. For example, a neoplastic or cancer cell can be distinguished from a normal cell by the uncontrolled growth and invasive properties characteristic of cancer cells. Using histological methods, a cancer cell can be observed to invade into surrounding normal tissue, have an increased mitotic index, and increased nuclear to cytoplasmic ratio, altered deposition of extracellular matrix, and a less differentiated phenotype. The unregulated proliferation of a cancer cell can be characterized by anchorage independent cell growth, proliferation in reduced-serum medium, loss of contact inhibition, and rapid proliferation compared to normal cells. Those skilled in the art will know how to determine if a TDP inhibiting compound is effective in promoting a more normal phenotype in a cancer cell. Those skilled in the art will also be able to detect a cancer cell in a population of cells, tumor, or organ.

[0099] Animal models of hyperproliferative diseases similarly can be used to assess the activity of TDP inhibiting compound or an amount sufficient to inhibit the activity of TDP in a cell. Animal models of such pathological conditions well known in the art which are reliable predictors of treatments in humans include, for example, animal models for tumor growth and metastasis and autoimmune disease.

[0100] Animal tumor models are known in the art which are predictive of the effects of therapeutic treatment. These models generally include the inoculation or implantation of a laboratory animal with heterologous tumor cells with simultaneous or subsequent administration of a therapeutic treatment. The efficacy of the treatment is determined by measuring the extent of tumor growth or metastasis. Measurement of clinical or physiological indicators can alternatively or additional be assessed as an indicator of treatment efficacy. Exemplary animal tumor models can be found described in, for example, Brugge et al. Origins of Human Cancer, Cold Spring Harbor Laboratory Press, Plain View, N.Y., (1991).

[0101] A variety of different classes of enzyme inhibitors can be used to inhibit TDP. For example, competitive inhibitors are designed to inhibit TDP by occupying the active site and preventing cleavage. Irreversible inhibitors are designed to place a chemically reactive group within the active site that can inactivate the enzyme. Mechanism-based inhibitors also generate reactive species within the active site, however the reactive groups are generated as a result of the cleavage reaction and should therefore be much more specific. Finally, small molecule inhibitors can be identified using a positional scanning method described herein.

[0102] One class of TDP inhibitors are nonhydrolyzable analogs. A nonhydrolyzable analog that binds tightly enough to the enzyme can function as a competitive inhibitor if the rate of disassociation (k_(off)) is sufficiently slow. Such analogs can be converted into nonhydrolyzable substrates by modifying the phosphodiester linkage between the DNA and the tyrosine residue (Wang et al., Med. Res. Rev. 17:367-425 (1997)). Bridging phosphoramidates have been demonstrated to be efficient nuclease inhibitors (Bannwarth, W. Helv. Chem. Acta 71:1517-1527 (1988)), and it has been shown that 5′-bridging phosphoramidate linkages prevent Topo I-mediated cleavage (Burgin, A., Huizenga, B., and Nash, H. Nuc. Acids Res. 23, 2973-2979 (1995)).

[0103] A second method for preventing phosphodiester hydrolysis and transesterification reactions is to substitute one of the non-bridging oxygens of the phosphodiester (Eckstein, F. Oligonucleotides and Their Analogs. (IRL Press) (1992)). Reagents useful for synthesizing phosphorothioate (Yang, S. et al., Proc. Natl. Acad. Sci. 93:11534-11539 (1996)) and methyl phosphonate analogs (Burgin, A. et al., Nuc. Acids Res. 23:2973-2979 (1995)) are commercially available (Glenn Research, Sterling, Virginia). These reagents can be conveniently used with tyrosine derivatized resins to yield the desired products. The phosphorothioate and phosphonate analogs can also be synthesized with a bridging phosphoramidate linkage to form a competitive inhibitor. A variety of different functionalities can be placed at the para position of the aromatic ring. For example, a chemically reactive group can be placed within the active site of the enzyme by positioning at the para position of the aromatic ring. If such a group reacts faster than the rate of dissociation, the inhibitor will become irreversibly linked to the enzyme. For example, an aziridine group placed at the para position places very reactive electrophile within the active site of the enzyme. The greatest difficulty with this approach is placing the aziridine functionality within a deprotected oligonucleotide. A strategy based on the method of convertible bases has been previously developed to place aziridine functions at the 4 position of thymidine (Zheng et al., Nucleos. Nucleot. 14:939-942 (1995)) within an oligonucleotide, and this technology can be applied to modify the 4 position (para) of 3′-phospho-phenyl oligonucleotides (see Example IV).

[0104] The third class of substrate analogs are designed to act as mechanism-based inhibitors. These analogs are similar to the irreversible inhibitors described above, however in this class of molecules a chemically reactive group is generated as a result of the cleavage reaction itself (Silverman, R. Mechanism-Based Enzyme inactivation: Chemistry and Enzymology (CRC Press) (1988)). This feature can dramatically increase the specificity of the inhibitor (see Example V).

[0105] The invention provides a method of screening for compounds that modulate the activity of TDP. The method comprises (a) contacting TDP, or a fragment or modification thereof having TDP activity, with a compound under conditions that allow TDP activity, (b) determining an amount of TDP activity, and (c) identifying a compound that modulates the activity of TDP, or a fragment of modification thereof.

[0106] A TDP to be contacted in the methods of the invention for screening for compounds that modulate the activity of TDP can be contained in a variety of sample types. For example, a TDP can be contained in an animal, including a human, such as when in vivo or in situ detection methods are employed, as well as in samples obtained or derived from the animal, such as when ex vivo detection methods are employed. A TDP can also be contained in a cell that recombinantly expresses TDP and in a lysate or fraction of such a cell. A TDP can be contained in a histologic section of a specimen obtained by biopsy, cells obtained from body fluids, or cells that are placed in or adapted to tissue culture. An isolated TDP is removed or separated from at least one component with which it is naturally associated. Therefore, an isolated TDP can be contained in a subcellular fraction or extract prepared from such cells, such as a cytoplasmic lysate, a membrane preparation, a nuclear extract, or a crude or purified protein preparation. A sample containing a TDP can be prepared by methods known in the art suitable for the particular format of the detection method. For example, biochemical methods such as precipitation, chromatography and immunoaffinity methods can be used to isolate a TDP from a cell which expresses TDP endogenously or recombinantly. Procedures for preparing subcellular fractions, such as nuclear fractions, and cell lysates are well known to those skilled in the art, and include, for example, cell disruption followed by separation methods such as gradient centrifugation and biochemical purification methods. An exemplary method for purifying TDP is described herein, in Example I.

[0107] An amount of TDP activity can be determined using a variety of methods. For example, the amount or rate of TDP activity can be determined using polynucleotide and non-polynucleotide substrates (see Example II). Exemplary assays for detecting 3′-tyrosyl-phosphodiesterase activity qualitatively and quantitatively are provided herein, in Example II. Other methods for determining phosphodiesterase activity well known to those skilled in the art can be used for determining an amount of TDP activity. The amount of TDP mRNA or polypeptide contained in a cell, tissue or organ indicates the amount of TDP activity present in the particular sample, and can therefore be used as a qualitative or quantitative indication of the amount of TDP activity in the sample. Methods for determining the amount of TDP mRNA and polypeptide amounts in a cell, tissue or organ, using in situ and in vitro methods well known to those skilled in the art can be used to determine an amount of TDP activity in a cell, tissue or organ.

[0108] As understood by those of skill in the art, assay methods for identifying compounds that modulate TDP activity generally require comparison to a control. An exemplary control is a cell or isolated TDP preparation that is treated substantially the same as the test cell or preparation exposed to a compound, except that the control is not exposed to the compound. A control cell or isolated TDP preparation can be treated with a carrier solution or solvent in which a compound is dissolved or contained, such as an aqueous or organic solution, if desired.

[0109] A compound identified using the methods of the invention can modulate TDP activity by a variety of mechanisms. For example, a compound can act directly by binding to TDP and altering a function, such as enzyme activity or substrate affinity or avidity, or can act indirectly by binding to a molecule that alters TDP activity. A molecule that alters TDP activity can function, for example, by modulating the amount of TDP expressed or contained in a cell. A compound can act to decrease TDP activity by decreasing the amount of TDP polypeptide in a cell, for example, by stimulating decreased TDP mRNA expression. TDP mRNA expression can be decreased, for example, by inducing or derepressing the transcription of a TDP gene and by regulating the expression of a cellular protein that acts as a transcription factor to regulate gene expression. A compound can act to decrease the amount of TDP activity by decreasing the stability of a TDP mRNA or polypeptide, for example, by increasing a cellular degradation activity, such as a protease activity. Conversely, a compound can act to increase an amount of TDP activity.

[0110] A TDP can be recombinantly expressed in a variety of cell types using well known expression systems and methods, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Plainview, N.Y. (2001)).

[0111] Compounds useful as potential therapeutic agents can be generated by methods well known to those skilled in the art, for example, well known methods for producing pluralities of compounds, including chemical or biological molecules such as simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources. Combinatorial libraries of molecules can be prepared using well known combinatorial chemistry methods (Gordon et al., J. Med. Chem. 37: 1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401 (1994); Gordon et al., Acc. Chem. Res. 29:144-154 (1996); Wilson and Czarnik, eds., Combinatorial Chemistry: Synthesis and Application, John Wiley & Sons, New York (1997)). Example VI provides an exemplary small molecule library which can be screened to identify a TDP modulating compound, and Example VII provides an exemplary method for deconvoluting such a small molecule library.

[0112] Such libraries can be screened to identify a compound that modulates TDP amount or activity using assay methods described herein, such as the spectrophotometric and fluorescence-based assays described in Example II. The effectiveness of compounds identified by an initial in vitro screen can be further tested in vivo using animal models of proliferative disease well known in the art. However, if desired, compounds can be screened using an in vivo assay.

[0113] Compounds identified as methods of the invention can be administered to an individual, for example, to alleviate a sign or symptom of a proliferative disease, such as cancer. Such compounds are useful therapeutic agents. One skilled in the art will know or can readily determine the alleviation of a sign or symptom associated with cancer, such as ovarian cancer, as methods for diagnosing and staging cancer are well known to those skilled in the art.

[0114] The invention provides a method of reducing the severity of a proliferative disease. The method involves administering to an individual an effective amount of a TDP inhibiting compound sufficient to inhibit the activity of TDP in cancer cells in the individual, thereby decreasing cell proliferation to reduce the severity of a proliferative disease.

[0115] The TDP modulating compounds of the invention can be formulated and administered by those skilled in the art in a manner and in an amount appropriate for the condition to be treated; the weight, gender, age and health of the individual; the biochemical nature, bioactivity, bioavailability and side effects of the particular compound; and in a manner compatible with concurrent treatment regimens. An appropriate amount and formulation for decreasing cell proliferation in humans can be extrapolated based on the activity of the compound in the assays described herein. An appropriate amount and formulation for use in humans for other indications can be extrapolated from credible animal models known in the art.

[0116] The total amount of compound can be administered as a single dose or by infusion over a relatively short period of time, or can be administered in multiple doses administered over a more prolonged period of time. Additionally, the compounds can be administered in slow-release matrices, which can be implanted for systemic delivery or at the site of the target tissue. Contemplated matrices useful for controlled release of therapeutic compounds are well known in the art, and include materials such as DepoFoam™, biopolymers, micropumps, and the like.

[0117] The compounds and compositions of the invention can be administered to the subject by any number of routes known in the art including, for example, intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intracisternally, intra-articularly, intracerebrally, orally, intravaginally, rectally, topically, intranasally, or transdermally. A preferred route for humans is oral administration.

[0118] A TDP modulating compound can be administered to a subject as a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. Those skilled in the art understand that the choice of a pharmaceutically acceptable carrier depends on the route of administration of the compound and on its particular physical and chemical characteristics. Pharmaceutically acceptable carriers are well known in the art and include sterile aqueous solvents such as physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils such as olive oil and injectable organic esters.

[0119] A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that stabilize the compound, increase its solubility, or increase its absorption. Such physiologically acceptable compounds include carbohydrates such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; and low molecular weight proteins.

[0120] For applications that require the compounds and compositions to cross the blood-brain barrier, formulations that increase the lipophilicity of the compound are particularly desirable. For example, a TDP modulating compound can be incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0121] In current cancer treatment, more than one compound is often administered to an individual for maximal reduction in symptoms. Thus, for use in treating cancer, a TDP modulating compound can be formulated with a second compound used for treating cancer. Compounds used for treating proliferative disease include, for example, cisplatin, flurouracil, methotrexate, ifosfamide, mitoxantrone, paclitaxel and camptothecins, such as Topotecan. Many of these drugs are administered to cancer patients at relatively high levels and adverse effects are common. Adverse side effects can be minimized by increasing the potency of a compound. Therefore, a TDP inhibiting compound can be combined with such compounds to effectively reduce symptoms in individuals having proliferative diseases such as cancer. A compound of the invention can be combined, for example, with a camptothecin. Types of camptothecins, formulations, routes of delivery and dosing of camptothecins are well known and are described, for example, in Kehrer et al., Anticancer Drugs, 12(2):89-105, (2001).

[0122] Camptothecin (CPT) family cytotoxic agents specifically inhibit topoisomerase I (Topo I). A number of experiments indicate that the actual target of camptothecins is the covalent 3′-phosphotyrosyl Topo I-DNA intermediate and that these poisons specifically inhibit the ligation step. First, the poisons do not appear to affect the rate of cleavage in vitro. However, CPT increases the yield of covalent intermediates when reactions are stopped with SDS. These results are consistent with a shift in the pseudo-equilibrium between cleavage and ligation because of a decrease in the rate of ligation. In addition, camptothecin and its derivatives bind to the covalent Topo I-DNA intermediate although the binding interactions are not well defined. Finally, CPT rapidly blocks both DNA and RNA synthesis in treated cells and is highly S-phase specific. Taken together, the data argues that stabilization of the covalent enzyme-DNA intermediate converts Topo I into a DNA damaging agent. Additional data demonstrates that this damage leads to the formation of persistent double strand breaks, that in turn trigger apoptosis or G-phase cell cycle arrest. Several other known Topo I poisons, including coralyne and Hoeschst 33342 also impede the religation step of catalysis and mediate cell killing by similar mechanisms. Cell killing by these compounds is caused by an accumulation of topoisomerase-DNA complexes which overwhelm the ability of the cell to repair the covalent complexes. Inhibition of the repair enzyme TDP will further increase the efficacy of such topoisomerase poisoning compounds in cell killing.

[0123] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLE I Purification of 3′-Tyrosyl-DNA-Phosphodiesterase

[0124] This example shows purification of calf thymus 3′-tyrosyl-DNA-phosphodiesterase.

[0125] 3′-tyrosyl-DNA-phosphodiesterase (TDP) was purified from calf thymus. A summary of the purification is provided in Table 1, below. The activity was difficult to detect after initial sonication and solubilization of the calf thymus but was detectable after the first purification step. The results demonstrate that the final preparation has a >300,000-fold greater specific activity over the starting crude lysate. Polyacrylamide gel analysis of the final enzyme preparation also indicates that a single dominant polypeptide is visible following Coomassie blue staining of the gel (two lanes from the same SDS polyacrylamide gel are shown in FIG. 1). Two fainter bands are present at approximately 60 and 65 KD. The data presented in Table 1 and FIG. 1 demonstrate that the current preparation is pure. TABLE 1 mg protein Units PDE* Units/mg Crude Lysate 910 5 0.005 Ion Exchange 3.1 360 120 Ion Exchange 0.5 550 1,100 Size Exclusion 0.3 400 1,600

[0126] Thus, TDP can be purified from a mammalian tissue using biochemical methods.

EXAMPLE II 3′-Tyrosyl-DNA-Phosphodiesterase Substrates and Activity Assays

[0127] This example shows TDP activity assays. DNA containing 5′-bridging phosphorothioate linkages are efficiently cleaved by Topoisomerase I. However, the cleavage generates a 5′-sulfhydryl (instead of a 5′-OH) that is an ineffective in subsequent ligation reactions thereby trapping the enzyme-DNA covalent intermediate (Burgin, et al. Nuc. Acids Res. 23:2973-2979 (1995)). TDP catalyzes the specific removal of topoisomerase I from this suicide DNA. Substrates containing only a single tyrosine linked to the 3′-end of DNA, were prepared. One type of substrate made use of a tyrosine residue present on a resin suitable for oligonucleotide synthesis. Different resins can be prepared in order to place different tyrosine analogs onto the 3′-end of the DNA. Following standard phosphoramidite synthesis and deprotection, a single tyrosine residue remained present on the 3′-end of the DNA. These substrates are cleaved efficiently by TDP.

[0128] Synthesis of competitive and irreversible inhibitors can be performed by placing different tyrosine analogs onto the 3′-end of DNA. Therefore, a post-synthetic approach for placing different analogs onto the 3′-end of DNA was developed. One substrate useful in a variety of convenient assay formats is 4-nitro-phenyl derivatized DNA. This substrate is particularly useful because cleavage by TDP generates 4-nitro-phenol, which absorbs at a unique wavelength (400 nm). This feature enables the assay to be detected spectrophotometrically. This is useful because the assays can be carried out in 96-well plates and the rate of cleavage can be measured in real time.

[0129] Oligonucleotides containing a 3′-monophosphate were synthesized. These oligonucleotides were purified and the 5′-dimethoxytrityl group was not removed in order to protect the 5′-OH. A water soluble condensing reagent (1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide; EDC) was then used to link 4-nitro-phenol to the 3′-phosphate group (Shabarova, Z. Biochimie 70:1323-1334 (1988)). The yield from this reaction was generally greater than 80%. In addition, the products were easily purified by reverse phase HPLC, as shown in FIG. 2. FIG. 2 shows that polynucleotide 17-mers labeled with EDC and 4-nitro-phenol can be detected by reverse phase HPLC. HPLC was performed using a Biorad Spherisorb column with 100 mM TEAA, 5-25% acetonitrile at 1 ml/min at 55° C. The underivatized polynucleotide had a retention time of 12.31 minutes (panel A). Incubation with EDC and 4-nitro-phenol caused the polynucleotide to elute at 13.15 minutes (panel B). A coinjection of underivatized and derivatized material demonstrated that the change in retention time is significant and reliable (panel C). This approach was used to derivatize polynucleotides with tyrosine, phenol, serine, aniline, and 4-nitro-aniline. These results demonstrate that a large number of different 3′-derivatized polynucleotides can be easily synthesized and purified.

[0130] The purified TDP does not cleave tyrosine analogs linked through a 5′-phosphodiester linkage. In addition, the enzyme does not degrade 5′-3′-DNA or -RNA phosphodiester linkages. Therefore, TDP activity was specific for 3′-tyrosyl-phosphodiesters. Similar results were observed for TDP purified from yeast (Yang, et al Proc. Natl. Acad. Sci. 93:11534-11539,(1996)).

[0131] TDP cleavage assays were detected by 5′-end labeling the 3′-derivatized oligonucleotides. In a typical reaction, TDP enzyme and oligonucleotides were incubated at 37° C. and then quenched with SDS (0.1%) and urea (5M final concentration). Reaction products were then resolved on a denaturing polyacrylamide sequencing gel (0.5×TBE, 0.5 mm gel, 15% acrylamide, 8M urea). The phosphodiesterase reaction generated an oligonucleotide containing a 3′phosphate, but lacking the 3′-tyrosine analog (4-nitro-phenol), which migrates faster within the gel than unreacted oligonucleotide. FIG. 3 shows the results oligonucleotide cleavage by TDP activity within individual fractions from a column purification. TDP activity was present in fractions 14-24.

[0132] The amount of cleavage can be accurately quantitated by phosphorimager analysis of a dried gel, allowing kinetic analysis of the enzyme. Reaction velocities were measured as a function of substrate concentration. In FIG. 4A, the reaction velocity (nM/min) was plotted as a function of substrate concentration (nM), resulting in a hyperbolic curve. The same data is plotted in FIG. 4B (Eadie-Hofstee plot), which indicates an apparent KM value of 1 mM.

[0133] The enzyme also efficiently cleaved a tyrosine analog when the substrate is duplex DNA. Data suggest that the tyrosine analog was cleaved efficiently (increased kcat/KM) when the substrate is present within a DNA duplex.

[0134] These results also demonstrate the ability to accurately measure small differences in reaction velocities/rates. This feature is useful for testing inhibitors.

[0135] TDP activity can be conveniently monitored by a variety of spectrophotometric and fluorescence-based assays. FIG. 9 shows the chemical structure of a TDP substrate useful for spectrophotometric detection of TDP activity. Cleavage of the substrate, shown on the left in FIG. 9, by TDP results in the formation of para-nitrophenol. Para-nitrophenyl absorbs UV light at 405 nm and as such, the amount of para-nitrophenyl present in a sample can be conveniently detected visually or using a spectrophotometer. A variety of substrates can be cleaved by TDP to form a detectable product. For example, a variety of substitutions can be made in the substrate shown in FIG. 9. In particular, the bridging oxygen in para-nitrophenyl can be replaced with sulfur or nitrogen, to generate 4-nitro-thiophenyl or 4-nitroaniline, shown below, which can be detected spectrophotometrically.

[0136]FIG. 10 shows the chemical structures of a TDP substrate useful in a fluorescence-based TDP activity assay, and the corresponding fluorescent product. In a TDP assay employing the substrate shown in FIG. 10, the para-nitrophenyl moiety acts as a quencher of a fluorescent base, etheno adenosine, which fluoresces upon excitation at about 300 nm. Prior to cleavage by TDP, the para-nitophenyl moiety absorbs 300 nm light and prevents maximal excitation of the etheno adenosine moiety. Upon TDP cleavage of the substrate, the para-nitrophenyl moiety is released from the substrate molecule, and fluorescence of the etheno adenosine moiety increases. Thus, TDP activity can be detected by measuring the formation of the fluorescent product. A variety of fluorescent bases or other detectable moieties can be incorporated into such a substrate at the position indicated in FIG. 10, as well as at additional or other positions within a substrate molecule. Additionally, a variety of moieties in addition to para-nitrophenyl can serve to quench the fluorescence of a selected fluorescent base or moiety.

[0137] Thus, TDP activity can be determined using a variety of polynucleotide and non-polynucleotide substrates, both qualitatively and quantitatively. Quantitative methods can be used to determine kinetic parameters for characterizing the ability of a compound to modulate TDP activity.

EXAMPLE III Synthesis of 3′-Tyrosine-DNA Phosphodiesterase Inhibitors

[0138] This example shows that TDP inhibitors can be synthesized via a condensation reaction using commercially availably reagents.

[0139] Polynucleotide derivative TDP inhibitors can be synthesized using the following methods. First, an oligonucleotide of 16 bases was synthesized on an ABI 392 DNA synthesizer using 3′-phosphate controlled pore glass resin (Glen Research, Sterling, Virginia) to build the DNA upon. The resultant oligo has a 3′ phosphate upon which the various functional groups are condensed. There are three different functional groups:

[0140] The Aniline derivative:

[0141] 100-200 uM of a 16 base oligonucleotide with a 3′ phosphate in 200 uL of water

[0142] 25 uL of MES at pH 5.5 as a buffer

[0143] 1 uL of 0.1 M MgCl

[0144] 22.8 uL of Aniline

[0145] 0.024 g of 1-[3-(Dimethylamino)propyl]-3ethylcarbodiimide hydrochloride 98+%

[0146] The above reagents were mixed in an eppendorf tube and shaken vigorously for 2 hours. The DNA was then precipitated using ethanol and 3M sodium acetate, and then re-suspended in 600 uL of water. The 600 uL re-suspension was then purified by high-pressure liquid chromatography via an anion exchange column (DIONEX).

[0147] The Toulidine derivative:

[0148] 100-200 uM of a 16 base oligonucleotide with a 3′ phosphate in 200 uL of water

[0149] 25 uL of MES at pH 5.5 as a buffer

[0150] 1 uL of 0.1 M MgCl

[0151] 0.0268 g of p-Toluidine

[0152] 0.024 g of 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride 98+%

[0153] The above reagents were mixed in an eppendorf tube. This mixture were warmed and vortexed briefly in order to get the p-Toluidine to go into solution (90° C. for 1 min). After the p-Toluidine dissolved, the mixture was shaken vigorously for 2 hours. The DNA was then precipitated using ethanol and 3M sodium acetate, and then re-suspended in 600 uL of water. The 600 uL re-suspension was then purified by high-pressure liquid chromatography via an anion exchange column (DIONEX).

[0154] The N′N-Dimethyl-1,4-phenylene-diamine derivative:

[0155] 100-200 uM of a 16 base oligonucleotide with a 3′ phosphate in 250 uL of water

[0156] 250 uL of acetonitrile

[0157] 25 uL of MES at pH 5.5 as a buffer

[0158] 1 uL of 0.1 M MgCl

[0159] 0.03405 of N′N-Dimethyl-1,4-phenylene-diamine 97%

[0160] 0.024 g of 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride 98+%

[0161] The above reagents were mixed in an eppendorf tube. This mixture had acteonitrile as one half the total volume since the N′N-Dimethyl-1,4-phenylene-diamine is not soluble in water. This mixture was shaken vigorously for 2 hours once everything was in solution. Two extractions were then done with acetonitrile. The DNA was then precipitated using ethanol and 3M sodium acetate, and then re-suspended in 600 uL of water. The 600 uL re-suspension was then purified by high-pressure liquid chromatography via an anion exchange column (DIONEX).

[0162] The Azidoaniline derivative:

[0163] 100-200 uM of a 16 base oligonucleotide with a 3′ phosphate in 200 uL of water

[0164] 25 uL of MES at pH 5.5 as a buffer

[0165] 1 uL of 0.1 M MgCl

[0166] 0.022 g of 4-Azidoaniline hydrochloride 97%

[0167] 0.024 g of 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride 98+%

[0168] The above reagents were mixed in an eppendorf tube, and the eppendorf tube was wrapped in aluminum foil to keep reduce the exposure to UV light. The tube was then shaken vigorously for 2 hours. The DNA was then precipitated using ethanol and 3M sodium acetate, and then re-suspended in 600 uL of water. The 600 uL re-suspension was then purified by high-pressure liquid chromatography via an anion exchange column (DIONEX).

[0169] Thus, 3-derivatized polynucleotides can be produced using commercially available reagents.

EXAMPLE IV Synthesis of Polynucleotides Containing a 3′ Phenyl Derivative

[0170] This example shows an exemplary method for synthesizing polynucleotides having 3′ phenyl derivatives.

[0171] In FIG. 5, a starting phosphoramidite is diagrammed on the left. The polynucleotides were synthesized in the non-standard 5′ to 3′ direction. The para position (“X” in FIG. 5) can be substituted with a functionality that can be replaced following synthesis and deprotection of the oligonucleotide. Following deprotection, an aziridine ion displaces X, resulting in an oligonucleotide containing a 3′-phospho-(p-azido)-phenyl group (Yeh, et al. J. Biol. Chem. 269, 15498-15405, (1994)). This method has also been used to place photo-reactive groups within oligonucleotides (Zheng, et al. Nucleos. Nucleot. 14, 939-942, (1995)). For example, an azido ion can be used to produce polynucleotides containing 3′-phospho-(p-aziridine)-phenyl groups (Lanza, et al., J. Biol. Chem. 271, 6978-6986, (1996)). For both of these inhibitors, it can be advantageous to prevent chemical reactions so that the enzyme-substrate complex is long-lived enough to allow crosslinking. This can be accomplished by synthesizing the azido and aziridine analogs with the bridging phosphoramidate linkages (Z═NH) described above.

[0172] Thus, a polynucleotide containing a 3′ phenyl derivative can be prepared by the described methods.

EXAMPLE V Mechanism-Based TDP Inhibitors

[0173] This example describes the rational for, and development of, mechanism-based TDP inhibitors.

[0174] The phosphodiesterase reaction generates DNA containing a 3′-phosphate and free tyrosine (Yang, S., et al. Proc. Natl. Acad. Sci. 93, 11534-11539, (1996)). Two potential mechanisms would result in these reaction products. In the simplest reaction, water (diagrammed as a hydroxide ion; for simplicity, individual proton donors and acceptors are not detailed in FIG. 6) attacks the phosphodiester bond displacing tyrosine. This simple hydrolysis reaction would result in DNA containing a 3′-phosphate and free tyrosine products. However, these same products can result from a two step reaction. In the first step, an enzyme-bound nucleophile (Nu: in FIG. 6) can attack the phosphodiester generating a covalent 3′-enzyme-DNA complex. Subsequent hydrolysis of this intermediate would result in the same products. This mechanism is similar to the mechanism used by tyrosine phosphatases. In the phosphatase reaction, the enzyme bound nucleophile can be serine, lysine, or cysteine. The 3′-phosphodiesterase activity and tyrosine phosphatases do have similar substrates: phosphodiester vs. phosphate linked to tyrosine. Therefore, the phosphodiesterase activity does not result from a simple hydrolysis reaction. Mechanism based inhibitors can be developed because both potential mechanisms rely upon an enzyme-bound hydrolysis step.

[0175] The compound 4-(fluoromethyl)phenyl phosphate (FMPP) has been demonstrated to be a potent inhibitor of prostatic acid phosphatase (PAP)(Myers, J., and Widlanski, T., Science 262:1451-1453 (1993)). In this reaction, PAP catalyzes the hydrolysis of the phosphate ester bond (rate constant k1 in FIG. 7) and leads to the formation of metastable phenoxide at the active site. This phenoxide eliminates a fluoride ion (ke_(elim)) to give a quinone methide, a very powerful alkylating agent, and can inactivate the enzyme through alkylation (k_(alk)) of an active site residue. Similar substrates have been used to study RNAse A (Stowell et al., J. Org. Chem. 60:6930-6936 (1995)) and calcinurin (Born et al., J. Biol. Chem. 270:25651-25655 (1995)).

[0176] It follows that DNA containing 3′-(4-(fluoromethyl)phenyl)-phosphate (3′-[FMPP]-DNA) can specifically alkylate TDP. The nucleophilic displacement of tyrosine (k₁) by water or some other nucleophile is analogous to the nucleophilic displacement of tyrosine during the phosphatase reaction; both are phosphoryl transfer reactions (SN2 nucleophilic displacement reactions) and in both cases the leaving group is tyrosine. The critical feature of this approach is the generation of a quinone methide at the active site of the enzyme and is therefore independent of the attacking nucleophile.

[0177] A feature of this approach is that the rate of fluoride elimination (k_(elim)) must be faster than the rate of dissociation (k_(diss1)); and the rate of alkylation (k_(alk)) must be faster than the rates of dissociation (k_(diss2)). This can only be determined experimentally. If the rate of elimination is too slow relative to dissociation (kdiss1), the reagent can be modified to increase this rate. For example, if the tyrosine bridging oxygen is replaced with sulfur, the rate of elimination will increase because the sulfhydryl will deprotonate faster leading to a faster formation of the quinone methide. This,modification should also increase the rate of cleavage (k₁) because sulfur-phosphorous bonds are less stable than oxygen-phosphorous bonds. The rate of alkylation is not easily manipulated because it will most likely be influenced by the positioning of an enzyme bound nucleophile (:Nu-Enz in FIG. 7). 3′-(FMP)-nucleotides can be synthesized, for example, as described in Stowell et al., J. Orq. Chem. 60:6930-6936 (1995). 3′-(FMP)-oligonucleotides can be synthesized using similar methods.

EXAMPLE VI Small Molecule Library Screening

[0178] This example describes the use of an exemplary small molecule library for screening to identify compounds that modulate TDP.

[0179] A small molecule library, such as the library shown in FIG. 8, can be screened using the methods described herein to identify TDP modulating compounds. In the compound library depicted in FIG. 8, a single phenyl ring can be derivatized with many different functional groups at three different positions (1, 2 or 3). If 10 different functional groups are placed at each of these positions, then 1000 different compounds will be present within the complete library. Such a library can have few compounds or several thousand compounds, depending on the desired properties of the compounds to be screened.

[0180] Two general approaches have been described for the identification of individual active compounds from synthetic libraries: iterative deconvolution vs. positional scanning (Ostresh,et al. Methods Enzymol. 267, 220-234 (1996)). Because the current TDP assay is sensitive, the positional scanning method can be used to deconvolute the combinatorial libraries. In this approach, sublibraries are constructed based on the number of different susbstituted positions. As shown in FIG. 9, three different sublibraries can be constructed to deconvolute the library represented in FIG. 8. Each sublibrary can be analyzed by synthesizing molecules with a defined functionality (R1-R10) at one position and a mixture of functional groups (Rx) at each of the other two positions. For example, sublibrary 1 can be analyzed by determining which functional group results in the most inhibition of TDP when position 2 and 3 are mixtures of all possible combinations. If 10 different functionalities are possible, then 10 different assays can be run in order to define which functionality results in the greatest inhibition. In other words, which functionality results in the greatest enzyme inhibition at position 1 when positions 2 and 3 are complete mixtures. As an illustration, the example in FIG. 9 defines functionality R5 to be most potent at position 1. The same analysis is then repeated at positions 2 and 3. In the example, functionality R7 is ideal at position 2 and R9 is ideal at position 3. From these results it is possible to identify the idealized inhibitor. In this example, the best inhibitor can be identified from a mixture of 1000 different compounds by simply performing 30 different assays.

[0181] A step in this process is identifying the best functionality at each diversity position. This can be accomplished by not only determining the amount enzyme inhibition, but the amount of inhibition as a function of inhibitor concentration (mixture concentration). The mixture that results in the greatest inhibition at the lowest concentration (IC50) will identify the best functionality at a given position.

[0182] Throughout this application various publications have been referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.

[0183] Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 171 <212> TYPE: PRT <213> ORGANISM: Drosophila melanogaster <400> SEQUENCE: 1 Asp Ser Thr Pro Val Gly Lys Leu Arg Gln Met Pro Pro Phe Lys Met 1 5 10 15 Ile Tyr Pro Ser Tyr Gly Asn Val Ala Gly Ser His Asp Gly Met Leu 20 25 30 Gly Gly Gly Cys Leu Pro Tyr Gly Lys Asn Thr Asn Asp Lys Gln Pro 35 40 45 Trp Leu Lys Asp Tyr Leu Gln Gln Trp Lys Ser Ser Asp Arg Phe Arg 50 55 60 Ser Arg Ala Met Pro His Ile Lys Ser Tyr Thr Arg Phe Asn Leu Glu 65 70 75 80 Asp Gln Ser Val Tyr Trp Phe Val Leu Thr Ser Ala Asn Leu Ser Lys 85 90 95 Ala Ala Trp Gly Cys Phe Asn Lys Asn Ser Asn Ile Gln Pro Cys Leu 100 105 110 Arg Ile Ala Asn Tyr Glu Ala Gly Val Leu Phe Leu Pro Arg Phe Val 115 120 125 Thr Gly Glu Asp Thr Phe Pro Leu Gly Asn Asn Arg Asp Gly Val Pro 130 135 140 Ala Phe Pro Leu Pro Tyr Asp Val Pro Leu Thr Pro Tyr Ala Pro Asp 145 150 155 160 Asp Lys Pro Phe Leu Met Asp Tyr Leu Gln Gly 165 170 <210> SEQ ID NO 2 <211> LENGTH: 319 <212> TYPE: PRT <213> ORGANISM: Mus musculus <400> SEQUENCE: 2 His Gln Lys Thr Gln Gly Ile Trp Leu Ser Pro Leu Tyr Pro Arg Ile 1 5 10 15 Asp Gln Gly Ser His Thr Ala Gly Glu Ser Ser Thr Arg Phe Lys Ala 20 25 30 Asp Leu Thr Ser Tyr Leu Thr Ala Tyr Asn Ala Pro Pro Leu Gln Glu 35 40 45 Trp Ile Asp Ile Ile Gln Glu His Asp Leu Ser Glu Thr Asn Val Tyr 50 55 60 Leu Ile Gly Ser Thr Pro Gly Arg Phe Gln Gly Ser His Arg Asp Asn 65 70 75 80 Trp Gly His Phe Arg Leu Arg Lys Leu Leu Gln Ala His Ala Pro Ser 85 90 95 Thr Pro Lys Gly Glu Cys Trp Pro Ile Val Gly Gln Phe Ser Ser Ile 100 105 110 Gly Ser Leu Gly Pro Asp Glu Ser Lys Trp Leu Cys Ser Glu Phe Lys 115 120 125 Asp Ser Leu Leu Ala Leu Arg Glu Glu Gly Arg Pro Pro Gly Lys Ser 130 135 140 Ala Val Pro Leu His Leu Ile Tyr Pro Ser Val Glu Asn Val Arg Thr 145 150 155 160 Ser Leu Glu Gly Tyr Pro Ala Gly Gly Ser Leu Pro Tyr Ser Ile Gln 165 170 175 Thr Ala Glu Lys Gln Arg Trp Leu His Ser Tyr Phe His Lys Trp Ser 180 185 190 Ala Glu Thr Ser Gly Arg Ser Asn Ala Met Pro His Ile Lys Thr Tyr 195 200 205 Met Arg Pro Ser Pro Asp Phe Ser Lys Leu Ala Trp Phe Leu Val Thr 210 215 220 Ser Ala Asn Leu Ser Lys Ala Ala Trp Gly Ala Leu Glu Lys Asn Gly 225 230 235 240 Thr Gln Leu Met Ile Arg Ser Tyr Glu Leu Gly Val Leu Phe Leu Pro 245 250 255 Ser Ala Phe Gly Leu Asp Thr Phe Lys Val Lys Gln Lys Phe Phe Ser 260 265 270 Ser Ser Cys Glu Pro Thr Ala Ser Phe Pro Val Pro Tyr Asp Leu Pro 275 280 285 Pro Glu Leu Tyr Gly Ser Lys Asp Arg Pro Trp Ile Trp Asn Ile Pro 290 295 300 Tyr Val Lys Ala Pro Asp Thr His Gly Asn Met Trp Val Pro Ser 305 310 315 <210> SEQ ID NO 3 <211> LENGTH: 264 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 <223> OTHER INFORMATION: Xaa = Any Amino Acid <400> SEQUENCE: 3 Glu Thr Asn Val Tyr Leu Ile Gly Ser Thr Pro Gly Arg Phe Gln Gly 1 5 10 15 Ser Gln Lys Asp Asn Trp Gly His Phe Arg Leu Lys Lys Leu Leu Lys 20 25 30 Asp His Ala Ser Ser Met Pro Asn Ala Glu Ser Trp Pro Val Val Gly 35 40 45 Gln Phe Ser Ser Val Gly Ser Leu Gly Ala Ser Glu Ser Lys Trp Leu 50 55 60 Cys Ser Glu Phe Lys Glu Ser Met Leu Thr Leu Gly Lys Glu Ser Lys 65 70 75 80 Thr Pro Gly Lys Ser Ser Val Pro Leu Tyr Leu Ile Tyr Pro Ser Val 85 90 95 Glu Asn Val Arg Thr Ser Leu Glu Gly Tyr Pro Ala Gly Gly Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Gln Asn Trp 115 120 125 Leu His Ser Tyr Phe His Lys Trp Ser Ala Glu Thr Ser Gly Arg Ser 130 135 140 Asn Ala Met Pro His Ile Lys Thr Tyr Met Arg Pro Ser Pro Asp Phe 145 150 155 160 Ser Lys Ile Ala Trp Phe Leu Val Thr Ser Ala Asn Leu Ser Lys Ala 165 170 175 Ala Trp Gly Ala Leu Glu Lys Asn Gly Thr Gln Leu Met Ile Arg Ser 180 185 190 Tyr Glu Leu Gly Val Leu Phe Leu Pro Ser Ala Phe Gly Leu Asp Ser 195 200 205 Phe Lys Val Lys Gln Lys Phe Phe Ala Gly Ser Gln Glu Pro Met Ala 210 215 220 Thr Phe Pro Val Pro Tyr Asp Leu Pro Pro Glu Leu Tyr Gly Ser Lys 225 230 235 240 Asp Arg Pro Trp Ile Trp Asn Ile Pro Tyr Val Lys Ala Pro Asp Thr 245 250 255 His Gly Asn Met Trp Val Pro Ser 260 <210> SEQ ID NO 4 <211> LENGTH: 343 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Leu Gly Trp Cys Leu Ser Ser Ser Asp Asp Glu Leu Gln Pro Glu Met 1 5 10 15 Pro Gln Lys Gln Ala Glu Lys Val Val Ile Lys Lys Glu Lys Asp Ile 20 25 30 Ser Ala Pro Asn Asp Gly Thr Ala Gln Arg Thr Glu Asn His Gly Ala 35 40 45 Pro Ala Cys His Arg Leu Lys Glu Glu Glu Asp Glu Tyr Glu Thr Ser 50 55 60 Gly Glu Gly Gln Asp Ile Trp Asp Met Leu Asp Lys Gly Asn Pro Phe 65 70 75 80 Gln Phe Tyr Leu Thr Arg Val Ser Gly Val Lys Pro Lys Tyr Asn Ser 85 90 95 Gly Ala Leu His Ile Lys Asp Ile Leu Ser Pro Leu Phe Gly Thr Leu 100 105 110 Val Ser Ser Ala Gln Phe Asn Tyr Cys Phe Asp Val Asp Trp Leu Val 115 120 125 Lys Gln Tyr Pro Pro Glu Phe Arg Lys Lys Pro Ile Leu Leu Val His 130 135 140 Gly Asp Lys Arg Glu Ala Lys Ala His Leu His Ala Gln Ala Lys Pro 145 150 155 160 Tyr Glu Asn Ile Ser Leu Cys Gln Ala Lys Leu Asp Ile Ala Phe Gly 165 170 175 Thr His His Thr Lys Met Met Leu Leu Leu Tyr Glu Glu Gly Leu Arg 180 185 190 Val Val Ile His Thr Ser Asn Leu Ile His Ala Asp Trp His Gln Lys 195 200 205 Thr Gln Gly Ile Trp Leu Ser Pro Leu Tyr Pro Arg Ile Ala Asp Gly 210 215 220 Thr His Lys Ser Gly Glu Ser Pro Thr His Phe Lys Ala Asp Leu Ile 225 230 235 240 Ser Tyr Leu Met Ala Tyr Asn Ala Pro Ser Leu Lys Glu Trp Ile Asp 245 250 255 Val Ile His Lys His Asp Leu Ser Glu Thr Asn Val Tyr Leu Ile Gly 260 265 270 Ser Thr Pro Gly Arg Phe Gln Gly Ser Gln Lys Asp Asn Trp Gly His 275 280 285 Phe Arg Leu Lys Lys Leu Leu Lys Asp His Ala Ser Ser Met Pro Asn 290 295 300 Ala Glu Ser Trp Pro Val Val Gly Gln Phe Ser Ser Val Gly Ser Leu 305 310 315 320 Gly Ala Asp Glu Ser Lys Trp Leu Cys Ser Glu Phe Lys Glu Ser Met 325 330 335 Leu Thr Leu Gly Lys Glu Ser 340 <210> SEQ ID NO 5 <211> LENGTH: 451 <212> TYPE: PRT <213> ORGANISM: Caenorhabditis elegans <400> SEQUENCE: 5 Met Lys Arg Thr Ile Gln Glu Thr Pro Gly Pro Ser Ser Thr Thr Val 1 5 10 15 Pro Pro Pro Lys Lys Leu Asn Ser Gln Arg Asn Gly Ser Asn Leu Glu 20 25 30 Pro Gly Ser Ile Tyr Phe Thr Pro Ile Gly Gly Ile Ser Val Pro Arg 35 40 45 Gln Glu Ser Glu Ser Ser Arg Ser Leu Asp Glu Ile Leu Ala Asp Ile 50 55 60 Arg Pro Ile Asn Ser Leu His Phe Ser Phe Met Leu Asp Phe Glu Phe 65 70 75 80 Leu Ile Gly Ser Tyr Pro Pro Ser Leu Arg Glu Tyr Pro Ile Thr Leu 85 90 95 Val Val Gly Ala Pro Asp Ala Pro Asp Leu Leu Lys Cys Thr Lys Asn 100 105 110 Gln Lys Leu Val Thr Val Val Gly Ala Ser Leu Pro Ile Pro Phe Gly 115 120 125 Thr His His Thr Lys Met Ser Ile Leu Glu Asp Glu Asp Gly Arg Phe 130 135 140 His Val Ile Val Ser Thr Ala Asn Leu Val Pro Asp Asp Trp Glu Phe 145 150 155 160 Lys Thr Gln Gln Phe Tyr Tyr Asn Phe Gly Val Lys Ile Ala Ser Gly 165 170 175 Thr Val Pro Arg Ser Asp Phe Gln Asp Asp Leu Leu Glu Tyr Leu Ser 180 185 190 Met Tyr Arg Asn Gln Leu Asp Thr Trp Lys Gln Leu Leu Gln Lys Val 195 200 205 Asp Phe Ser Gln Ile Ser Asp Arg Leu Ile Phe Ser Thr Pro Gly Tyr 210 215 220 His Thr Asp Pro Pro Thr Gln Arg Pro Gly His Pro Arg Leu Phe Arg 225 230 235 240 Ile Leu Ser Glu Lys Phe Pro Phe Asp Ala Ser Tyr Glu His Thr Glu 245 250 255 Arg Cys Thr Phe Val Ala Gln Cys Ser Ser Ile Gly Ser Leu Gly Ser 260 265 270 Ala Pro Ile Asn Trp Phe Arg Gly Gln Phe Leu Gln Ser Leu Glu Gly 275 280 285 Ala Asn Pro Ser Pro Lys Gln Lys Pro Ala Lys Met Tyr Leu Val Phe 290 295 300 Pro Ser Val Glu Asp Val Arg Thr Ser Cys Gln Gly Tyr Ala Gly Gly 305 310 315 320 Cys Ser Val Pro Tyr Arg Asn Ser Val His Ala Arg Gln Lys Trp Leu 325 330 335 Gln Gly Asn Met Cys Lys Trp Arg Ser Asn Ala Lys Arg Arg Thr Asn 340 345 350 Ala Val Pro His Cys Lys Thr Tyr Val Lys Tyr Asp Lys Lys Val Ala 355 360 365 Ile Trp Gln Leu Leu Thr Ser Ala Asn Leu Ser Lys Ala Ala Trp Gly 370 375 380 Glu Val Ser Phe Asn Lys Ser Lys Asn Val Glu Gln Leu Met Ile Arg 385 390 395 400 Ser Trp Glu Met Gly Val Leu Ile Thr Asp Pro Ser Arg Phe Asn Ile 405 410 415 Pro Phe Asp Tyr Pro Leu Val Pro Tyr Ser Ala Thr Asp Glu Pro Phe 420 425 430 Val Thr Asp Lys Lys His Glu Lys Pro Asp Ile Leu Gly Cys Ile Trp 435 440 445 Thr Pro Pro 450 <210> SEQ ID NO 6 <211> LENGTH: 544 <212> TYPE: PRT <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 6 Met Ser Arg Glu Thr Asn Phe Asn Gly Thr Lys Arg Lys Arg Ser Asp 1 5 10 15 Val Ala Glu Lys Val Ala Gln Arg Trp Lys Ser Val Arg Tyr Ser Ala 20 25 30 Glu Met Glu Asn Met Ala Pro Val Asn Ser Asn Asn Asp Ser Asp Asp 35 40 45 Cys Val Ile Val Ser Glu Ser Lys Ile Ile Asp Leu Thr Asn Gln Glu 50 55 60 Gln Asp Leu Ser Glu Arg Ile Glu Thr Asn Asp Thr Ala Lys Gly Ala 65 70 75 80 Val Phe Lys Leu Met Lys Ser Asp Phe Tyr Glu Arg Glu Asp Phe Met 85 90 95 Gly Glu Val Glu Asp Met Ile Thr Leu Lys Asp Ile Phe Gly Thr Glu 100 105 110 Thr Leu Lys Arg Ser Ile Leu Phe Ser Phe Gln Tyr Glu Leu Asp Phe 115 120 125 Leu Leu Arg Gln Phe His Gln Asn Val Glu Asn Ile Thr Ile Val Gly 130 135 140 Gln Lys Gly Thr Ile Met Pro Ile Glu Ala Arg Ala Met Asp Ala Thr 145 150 155 160 Leu Ala Val Ile Leu Lys Lys Val Lys Leu Ile Glu Ile Thr Met Pro 165 170 175 Pro Phe Ala Ser His His Thr Lys Leu Ile Ile Asn Phe Tyr Asp Asn 180 185 190 Gly Glu Cys Lys Ile Phe Leu Pro Ser Asn Asn Phe Thr Ser Met Glu 195 200 205 Thr Asn Leu Pro Gln Gln Val Cys Trp Cys Ser Pro Leu Leu Lys Ile 210 215 220 Gly Lys Glu Gly Leu Pro Val Pro Phe Lys Arg Ser Leu Ile Glu Tyr 225 230 235 240 Leu Asn Ser Tyr His Leu Lys Asp Ile Asp Glu Leu Ile Thr Lys Ser 245 250 255 Val Glu Glu Val Asn Phe Ala Pro Leu Ser Glu Leu Glu Phe Val Tyr 260 265 270 Ser Thr Pro Ser Lys Phe Gln Ser Ser Gly Leu Leu Ser Phe Tyr Asn 275 280 285 Lys Leu Glu Lys Leu Ser Ala Gly Thr Ser Ala Ser Asp Thr Ala Lys 290 295 300 His Tyr Leu Cys Gln Thr Ser Ser Ile Gly Thr Ser Leu Ser Arg Ala 305 310 315 320 Arg Asp Glu Asn Leu Trp Thr His Leu Met Ile Pro Leu Phe Thr Gly 325 330 335 Ile Met Ser Pro Pro Ala Lys Asp Thr Ala Gly Arg Lys Lys Ala Glu 340 345 350 Ile Leu Pro Thr Asn Ser Leu Ile Asn Glu Tyr Ser Gln Arg Lys Ile 355 360 365 Lys Pro Tyr Ile Ile Phe Pro Thr Glu Gln Glu Phe Val Thr Ser Pro 370 375 380 Leu Lys Trp Ser Ser Ser Gly Trp Phe His Phe Gln Tyr Leu Gln Lys 385 390 395 400 Lys Ser Tyr Tyr Glu Met Leu Arg Asn Lys Phe Lys Val Phe Tyr Lys 405 410 415 Gln Asp Pro Ala Met Val Thr Arg Arg Arg Gly Thr Thr Pro Ala His 420 425 430 Ser Lys Phe Tyr Met His Cys Ala Thr Asn Ser Ala Gly Pro Cys Asp 435 440 445 Ala Ser Gln Val Phe Lys Glu Leu Glu Trp Cys Leu Tyr Thr Ser Ala 450 455 460 Asn Leu Ser Gln Thr Ala Trp Gly Thr Val Ser Arg Lys Pro Arg Asn 465 470 475 480 Tyr Glu Ala Gly Val Leu Tyr His Ser Arg Arg Leu Ala Asn Thr Arg 485 490 495 Lys Val Thr Cys Arg Thr Phe Thr Arg Asp Arg Arg Gly Cys Ala Gly 500 505 510 Asn Pro Thr His Val Ala Val Pro Phe Thr Leu Pro Val Ile Pro Tyr 515 520 525 Asp Leu Ala Glu Asp Glu Cys Phe Cys Leu Ala Arg His Glu Asn Asp 530 535 540 <210> SEQ ID NO 7 <211> LENGTH: 2344 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (251)...(2074) <400> SEQUENCE: 7 gttggttctg tgcgcctcag agttggagca cacagctgta ttaaaaaggc aaatcgaagg 60 ccgggcgcgg tgactcacgc ctgtcatcct agcactttgg gaggccgagg cggctgaatc 120 acttgaggtt aggagtttga gatcagcccg ggcaacatgg tgaaaccccg tctctacaaa 180 aatagaaaaa ttagccgagc gtgatggtgg atgcctgtaa tcctagctcc tcgggaggct 240 aaggagtata atg tct cag gaa ggc gat tat ggg agg tgg acc ata tct 289 Met Ser Gln Glu Gly Asp Tyr Gly Arg Trp Thr Ile Ser 1 5 10 agt agt gat gaa agt gag gaa gaa aag cca aaa cca gac aag cca tct 337 Ser Ser Asp Glu Ser Glu Glu Glu Lys Pro Lys Pro Asp Lys Pro Ser 15 20 25 acc tct tct ctt ctc tgt gcc agg caa gga gca gca aat gag ccc agg 385 Thr Ser Ser Leu Leu Cys Ala Arg Gln Gly Ala Ala Asn Glu Pro Arg 30 35 40 45 tac acc tgt tcc gag gcc cag aaa gct gca cac aag agg aaa ata tca 433 Tyr Thr Cys Ser Glu Ala Gln Lys Ala Ala His Lys Arg Lys Ile Ser 50 55 60 cct gtg aaa ttc agc aat aca gat tca gtt tta cct ccc aaa agg cag 481 Pro Val Lys Phe Ser Asn Thr Asp Ser Val Leu Pro Pro Lys Arg Gln 65 70 75 aaa agc ggt tcc cag gag gac ctc ggc tgg tgt ctg tcc agc agt gat 529 Lys Ser Gly Ser Gln Glu Asp Leu Gly Trp Cys Leu Ser Ser Ser Asp 80 85 90 gat gag ctg caa cca gaa atg ccg cag aag cag gct gag aaa gtg gtg 577 Asp Glu Leu Gln Pro Glu Met Pro Gln Lys Gln Ala Glu Lys Val Val 95 100 105 atc aaa aag gag aaa gac atc tct gct ccc aat gac ggc act gcc caa 625 Ile Lys Lys Glu Lys Asp Ile Ser Ala Pro Asn Asp Gly Thr Ala Gln 110 115 120 125 aga act gaa aat cat ggc gct ccc gcc tgc cac agg ctc aaa gag gag 673 Arg Thr Glu Asn His Gly Ala Pro Ala Cys His Arg Leu Lys Glu Glu 130 135 140 gaa gac gag tat gag aca tca ggg gag ggc cag gac att tgg gac atg 721 Glu Asp Glu Tyr Glu Thr Ser Gly Glu Gly Gln Asp Ile Trp Asp Met 145 150 155 ctg gat aaa ggg aac ccc ttc cag ttt tac ctc act aga gtc tct gga 769 Leu Asp Lys Gly Asn Pro Phe Gln Phe Tyr Leu Thr Arg Val Ser Gly 160 165 170 gtt aag cca aag tat aac tct gga gcc ctc cac atc aag gat att tta 817 Val Lys Pro Lys Tyr Asn Ser Gly Ala Leu His Ile Lys Asp Ile Leu 175 180 185 tct cct tta ttt ggg acg ctt gtt tct tca gct cag ttt aac tac tgc 865 Ser Pro Leu Phe Gly Thr Leu Val Ser Ser Ala Gln Phe Asn Tyr Cys 190 195 200 205 ttt gac gtg gac tgg ctc gta aaa cag tat cca cca gag ttc agg aag 913 Phe Asp Val Asp Trp Leu Val Lys Gln Tyr Pro Pro Glu Phe Arg Lys 210 215 220 aag cca atc ctg ctt gtg cat ggt gat aag cga gag gct aag gct cac 961 Lys Pro Ile Leu Leu Val His Gly Asp Lys Arg Glu Ala Lys Ala His 225 230 235 ctc cat gcc cag gcc aag cct tac gag aac atc tct ctc tgc cag gca 1 009 Leu His Ala Gln Ala Lys Pro Tyr Glu Asn Ile Ser Leu Cys Gln Ala 240 245 250 aag ttg gat att gcg ttt gga aca cac cac acg aaa atg atg ctg ctg 1 057 Lys Leu Asp Ile Ala Phe Gly Thr His His Thr Lys Met Met Leu Leu 255 260 265 ctc tat gaa gaa ggc ctc cgg gtt gtc ata cac acc tcc aac ctc atc 1105 Leu Tyr Glu Glu Gly Leu Arg Val Val Ile His Thr Ser Asn Leu Ile 270 275 280 285 cat gct gac tgg cac cag aaa act caa gga ata tgg ttg agc ccc tta 1153 His Ala Asp Trp His Gln Lys Thr Gln Gly Ile Trp Leu Ser Pro Leu 290 295 300 tac cca cga att gct gat gga acc cac aaa tct gga gag tcg cca aca 1201 Tyr Pro Arg Ile Ala Asp Gly Thr His Lys Ser Gly Glu Ser Pro Thr 305 310 315 cat ttt aaa gct gat ctc atc agt tac ttg atg gct tat aat gcc cct 1249 His Phe Lys Ala Asp Leu Ile Ser Tyr Leu Met Ala Tyr Asn Ala Pro 320 325 330 tct ctc aag gag tgg ata gat gtc att cac aag cac gat ctc tct gaa 1297 Ser Leu Lys Glu Trp Ile Asp Val Ile His Lys His Asp Leu Ser Glu 335 340 345 aca aat gtt tat ctt att ggt tca acc cca gga cgc ttt caa gga agt 1345 Thr Asn Val Tyr Leu Ile Gly Ser Thr Pro Gly Arg Phe Gln Gly Ser 350 355 360 365 caa aaa gat aat tgg gga cat ttt aga ctt aag aag ctt ctg aaa gac 1393 Gln Lys Asp Asn Trp Gly His Phe Arg Leu Lys Lys Leu Leu Lys Asp 370 375 380 cat gcc tca tcc atg cct aac gca gag tcc tgg cct gtc gta ggt cag 1441 His Ala Ser Ser Met Pro Asn Ala Glu Ser Trp Pro Val Val Gly Gln 385 390 395 ttt tca agc gtt ggc tcc ttg gga gcc gat gaa tca aag tgg tta tgt 1489 Phe Ser Ser Val Gly Ser Leu Gly Ala Asp Glu Ser Lys Trp Leu Cys 400 405 410 tct gag ttt aaa gag agc atg ctg aca ctg ggg aag gaa agc aag act 1537 Ser Glu Phe Lys Glu Ser Met Leu Thr Leu Gly Lys Glu Ser Lys Thr 415 420 425 cca gga aaa agc tct gtt cct ctt tac ttg atc tat cct tct gtg gaa 1585 Pro Gly Lys Ser Ser Val Pro Leu Tyr Leu Ile Tyr Pro Ser Val Glu 430 435 440 445 aat gtg cgg acc agt tta gaa gga tat cct gct ggg ggc tct ctt ccc 1633 Asn Val Arg Thr Ser Leu Glu Gly Tyr Pro Ala Gly Gly Ser Leu Pro 450 455 460 tat agc atc cag aca gct gaa aaa cag aat tgg ctg cat tcc tat ttt 1681 Tyr Ser Ile Gln Thr Ala Glu Lys Gln Asn Trp Leu His Ser Tyr Phe 465 470 475 cac aaa tgg tca gct gag act tct ggc cgc agc aat gcc atg cca cat 1729 His Lys Trp Ser Ala Glu Thr Ser Gly Arg Ser Asn Ala Met Pro His 480 485 490 att aag aca tat atg agg cct tct cca gac ttc agt aaa att gct tgg 1777 Ile Lys Thr Tyr Met Arg Pro Ser Pro Asp Phe Ser Lys Ile Ala Trp 495 500 505 ttc ctt gtc aca agc gca aat ctg tcc aag gct gcc tgg gga gca ttg 1825 Phe Leu Val Thr Ser Ala Asn Leu Ser Lys Ala Ala Trp Gly Ala Leu 510 515 520 525 gag aag aat ggc acc cag ctg atg atc cgc tcc tac gag ctc ggg gtc 1873 Glu Lys Asn Gly Thr Gln Leu Met Ile Arg Ser Tyr Glu Leu Gly Val 530 535 540 ctt ttc ctc cct tca gca ttt ggt cta gac agt ttc aaa gtg aaa cag 1921 Leu Phe Leu Pro Ser Ala Phe Gly Leu Asp Ser Phe Lys Val Lys Gln 545 550 555 aag ttc ttc gct ggc agc cag gag cca atg gcc acc ttt cct gtg cca 1969 Lys Phe Phe Ala Gly Ser Gln Glu Pro Met Ala Thr Phe Pro Val Pro 560 565 570 tat gat ttg cct cca gaa ctg tat gga agt aaa gat cgg cca tgg ata 2017 Tyr Asp Leu Pro Pro Glu Leu Tyr Gly Ser Lys Asp Arg Pro Trp Ile 575 580 585 tgg aac att cct tat gtc aaa gca ccg gat acg cat ggg aac atg tgg 2065 Trp Asn Ile Pro Tyr Val Lys Ala Pro Asp Thr His Gly Asn Met Trp 590 595 600 605 gtg ccc tcc tgagaatctt gaggcactgt gaaatttaag tgtaagacat 2114 Val Pro Ser tgagccacaa acatggaatc tcttctttgt actggatgtc cacttccctt aaagtcttat 2174 ttgcaccctt acaaaatctt tccaaaggtc actcttatga atggatgttg gttatacttt 2234 taatggacat taacattcct aataaagtat tagtttctta aaaaaaaaaa aaaaaaaaaa 2294 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2344 <210> SEQ ID NO 8 <211> LENGTH: 608 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 8 Met Ser Gln Glu Gly Asp Tyr Gly Arg Trp Thr Ile Ser Ser Ser Asp 1 5 10 15 Glu Ser Glu Glu Glu Lys Pro Lys Pro Asp Lys Pro Ser Thr Ser Ser 20 25 30 Leu Leu Cys Ala Arg Gln Gly Ala Ala Asn Glu Pro Arg Tyr Thr Cys 35 40 45 Ser Glu Ala Gln Lys Ala Ala His Lys Arg Lys Ile Ser Pro Val Lys 50 55 60 Phe Ser Asn Thr Asp Ser Val Leu Pro Pro Lys Arg Gln Lys Ser Gly 65 70 75 80 Ser Gln Glu Asp Leu Gly Trp Cys Leu Ser Ser Ser Asp Asp Glu Leu 85 90 95 Gln Pro Glu Met Pro Gln Lys Gln Ala Glu Lys Val Val Ile Lys Lys 100 105 110 Glu Lys Asp Ile Ser Ala Pro Asn Asp Gly Thr Ala Gln Arg Thr Glu 115 120 125 Asn His Gly Ala Pro Ala Cys His Arg Leu Lys Glu Glu Glu Asp Glu 130 135 140 Tyr Glu Thr Ser Gly Glu Gly Gln Asp Ile Trp Asp Met Leu Asp Lys 145 150 155 160 Gly Asn Pro Phe Gln Phe Tyr Leu Thr Arg Val Ser Gly Val Lys Pro 165 170 175 Lys Tyr Asn Ser Gly Ala Leu His Ile Lys Asp Ile Leu Ser Pro Leu 180 185 190 Phe Gly Thr Leu Val Ser Ser Ala Gln Phe Asn Tyr Cys Phe Asp Val 195 200 205 Asp Trp Leu Val Lys Gln Tyr Pro Pro Glu Phe Arg Lys Lys Pro Ile 210 215 220 Leu Leu Val His Gly Asp Lys Arg Glu Ala Lys Ala His Leu His Ala 225 230 235 240 Gln Ala Lys Pro Tyr Glu Asn Ile Ser Leu Cys Gln Ala Lys Leu Asp 245 250 255 Ile Ala Phe Gly Thr His His Thr Lys Met Met Leu Leu Leu Tyr Glu 260 265 270 Glu Gly Leu Arg Val Val Ile His Thr Ser Asn Leu Ile His Ala Asp 275 280 285 Trp His Gln Lys Thr Gln Gly Ile Trp Leu Ser Pro Leu Tyr Pro Arg 290 295 300 Ile Ala Asp Gly Thr His Lys Ser Gly Glu Ser Pro Thr His Phe Lys 305 310 315 320 Ala Asp Leu Ile Ser Tyr Leu Met Ala Tyr Asn Ala Pro Ser Leu Lys 325 330 335 Glu Trp Ile Asp Val Ile His Lys His Asp Leu Ser Glu Thr Asn Val 340 345 350 Tyr Leu Ile Gly Ser Thr Pro Gly Arg Phe Gln Gly Ser Gln Lys Asp 355 360 365 Asn Trp Gly His Phe Arg Leu Lys Lys Leu Leu Lys Asp His Ala Ser 370 375 380 Ser Met Pro Asn Ala Glu Ser Trp Pro Val Val Gly Gln Phe Ser Ser 385 390 395 400 Val Gly Ser Leu Gly Ala Asp Glu Ser Lys Trp Leu Cys Ser Glu Phe 405 410 415 Lys Glu Ser Met Leu Thr Leu Gly Lys Glu Ser Lys Thr Pro Gly Lys 420 425 430 Ser Ser Val Pro Leu Tyr Leu Ile Tyr Pro Ser Val Glu Asn Val Arg 435 440 445 Thr Ser Leu Glu Gly Tyr Pro Ala Gly Gly Ser Leu Pro Tyr Ser Ile 450 455 460 Gln Thr Ala Glu Lys Gln Asn Trp Leu His Ser Tyr Phe His Lys Trp 465 470 475 480 Ser Ala Glu Thr Ser Gly Arg Ser Asn Ala Met Pro His Ile Lys Thr 485 490 495 Tyr Met Arg Pro Ser Pro Asp Phe Ser Lys Ile Ala Trp Phe Leu Val 500 505 510 Thr Ser Ala Asn Leu Ser Lys Ala Ala Trp Gly Ala Leu Glu Lys Asn 515 520 525 Gly Thr Gln Leu Met Ile Arg Ser Tyr Glu Leu Gly Val Leu Phe Leu 530 535 540 Pro Ser Ala Phe Gly Leu Asp Ser Phe Lys Val Lys Gln Lys Phe Phe 545 550 555 560 Ala Gly Ser Gln Glu Pro Met Ala Thr Phe Pro Val Pro Tyr Asp Leu 565 570 575 Pro Pro Glu Leu Tyr Gly Ser Lys Asp Arg Pro Trp Ile Trp Asn Ile 580 585 590 Pro Tyr Val Lys Ala Pro Asp Thr His Gly Asn Met Trp Val Pro Ser 595 600 605 

What is claimed is:
 1. A compound of the formula:

wherein, X is a nucleotide; y is a positive integer; R1 is any nucleotide base; R2 is H; and R3 is phenyl or substituted phenyl.
 2. The compound of claim 1, wherein R2 is H.
 3. The compound of claim 2, wherein R3 is phenyl.
 4. A composition, comprising the compound of claim 1 and a camptothecin.
 5. A method of inhibiting 3′-tyrosyl-DNA phophodiesterase (TDP) activity, comprising contacting a TDP with a compound of claim
 1. 6. A method of decreasing cellular proliferation, comprising contacting a TDP-containing cell with an effective amount of a TDP inhibiting compound sufficient to inhibit TDP activity in said cell.
 7. The method of claim 6, wherein said TDP inhibiting compound is selected from the group consisting of polynucleotide-3′-bridging phosphoramidate, polynucleotide-3′-alkyl phosphonate, polynucleotide-3′-alkyl phosphotriester, polynucleotide-bridging-alkyl phosphonate, nucleotide-3′-bridging phosphoramidate, nucleotide-3′-alkyl phosphonate, nucleotide-3′-alkyl phosphotriester, and nucleotide-bridging-alkyl phosphonate.
 8. The method of claim 6, wherein said cell is a cancer cell.
 9. The method of claim 8, wherein said cancer cell is a cancer cell selected from the group consisting of ovarian, small-cell, breast, leukemia and lymphoma cancer cell.
 10. A method of reducing the severity of a proliferative disease, comprising administering to an individual an effective amount of a TDP inhibiting compound sufficient to inhibit the activity of TDP in a cell in said individual, thereby decreasing cell proliferation to reduce the severity of said proliferative disease.
 11. The method of claim 10, wherein said TDP inhibiting compound is selected from the group consisting of polynucleotide-3′-bridging phosphoramidate, polynucleotide-3′-alkyl phosphonate, polynucleotide-3′-alkyl phosphotriester, polynucleotide-bridging-alkyl phosphonate, nucleotide-3′-bridging phosphoramidate, nucleotide-3′-alkyl phosphonate, nucleotide-3′-alkyl phosphotriester, and nucleotide-bridging-alkyl phosphonate.
 12. The method of claim 10, wherein said proliferative disease is a cancer selected from the group consisting of ovarian cancer, small-cell cancer, breast cancer, leukemia and lymphoma.
 13. A method of screening for compounds that modulate the activity of TDP, comprising: (a) contacting TDP, or a fragment or modification thereof having TDP activity, with a compound under conditions that allow TDP activity; (b) determining an amount of TDP activity; and (c) identifying a compound that modulates TDP activity. 